Composition containing acetobacter saxatilis and nitrogen-fixing pantoea and application of composition

Abstract

The invention provides a composition containing acetobacter saxatilis and pantoea azototrophicus and application of the composition. The composition provided by the invention comprises acetobacter sativus or progeny thereof and pantoea azototrichum or progeny thereof; the registration number of the acetobacter breviscapus in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No.36149; the registration number of the nitrogen-fixing pantoea in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No.36150. The invention further discloses a preparation method of the nitrogen-fixing pantoea. The acetobacter sativus and the nitrogen-fixing pantoea or the composition of the acetobacter sativus and the nitrogen-fixing pantoea can be used for reducing emission of nitrogen-containing gas in the livestock breeding process and the livestock manure (normal state and composting) treatment process, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to compositions comprising Acetobacter oryzae and Azotobacter nigra and their applications. Background Technology

[0002] Livestock farming is a fundamental industry that ensures the supply of high-quality protein for humankind and maintains the vitality of rural economies and the stability of related industrial chains. However, it also generates large amounts of nitrogen-containing gases (ammonia, nitrous oxide, nitrogen oxides, etc.). Inside livestock sheds, high concentrations of ammonia can directly damage the respiratory mucosa of livestock and poultry, inducing diseases, reducing growth performance and immunity, seriously harming animal welfare and production efficiency, and threatening the health of workers. Once released into the environment, these gases become significant contributors to smog, acid rain, and eutrophication of water bodies, while nitrous oxide is a potent greenhouse gas, exacerbating global climate change. Therefore, effectively controlling nitrogen-containing gas emissions is a key challenge connecting the sustainable development of livestock farming with environmental protection. Given that nitrogen-containing gases are usually mixtures of multiple gases, the effectiveness of single bacterial strains in practical applications is limited; therefore, the development of compound microbial agents is particularly important and urgent. Summary of the Invention

[0003] The main problem this invention aims to solve is how to remove various nitrogen-containing gases produced during livestock farming.

[0004] In a first aspect, the present invention provides a composition comprising Acetobacter oryzae ( Acetobacter oryzae ) or their offspring and nitrogen-fixing bacteria ( Azoarcus sp.) or its descendants; The Acetobacter oryzae is deposited at the China General Microbiological Culture Collection Center (CGMCC), and its registration number at CGMCC is CGMCC No. 36149. The *Azotocinobacter* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), and its registration number at CGMCC is CGMCC No. 36150.

[0005] As mentioned above, the progeny of Acetobacter oryzae / Azotocinobacter refers to daughter cells produced through the growth of microorganisms (e.g., culture in a culture medium). It is understood that the progeny of Acetobacter oryzae / Azotocinobacter still retain the functions of Acetobacter oryzae / Azotocinobacter.

[0006] The composition described above is a microbial agent.

[0007] The composition described above may also contain other biological or non-biological components. Other active ingredients of the above-mentioned bacterial agent can be determined by those skilled in the art based on their effect on nitrogen-containing gas removal.

[0008] The composition described above further includes a carrier. The carrier can be a biologically inert carrier commonly used in environmental remediation. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one selected from clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one selected from corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0009] In one specific embodiment, the carrier is a solid carrier, which includes biochar, potassium humate, wheat bran, calcium carbonate, and diatomaceous earth; wherein the biochar accounts for 50% by mass of the solid microbial inoculant, the potassium humate accounts for 14% by mass of the solid microbial inoculant, the wheat bran accounts for 20% by mass of the solid microbial inoculant, the calcium carbonate accounts for 3% by mass of the solid microbial inoculant, and the diatomaceous earth accounts for 7% by mass of the solid microbial inoculant.

[0010] The composition as described above, based on each gram of the composition, contains 1.0 × 10⁻⁶. 8 -8.0×10 8 CFU of Acetobacter oryzae or its progeny and 1.0 × 10 9 -1.0×10 10 CFU of *Azotocinus* or its progeny. Further, based on each gram of the composition, the composition contains 1.4 × 10⁻⁶. 8 CFU of Acetobacter oryzae or its progeny and 2.0 × 10 9 CFU (Cytoxobacterium nitrificum) or its progeny.

[0011] Secondly, the present invention provides Acetobacter oryzae or its descendants, wherein the Acetobacter oryzae is deposited at the China General Microbiological Culture Collection Center (CGMCC) and the registration number of the Acetobacter oryzae at CGMCC is CGMCC No. 36149.

[0012] In a third aspect, the present application provides a nitrogen-fixing Arcobacter, which is deposited with the China General Microbiological Culture Collection Center (CGMCC) and has the accession number of CGMCC No. 36150.

[0013] In a fourth aspect, the present application provides a preparation method of a microbial agent, which comprises the step of preparing a microbial agent from the above composition, the above A. shizayakii or its descendant, or the above nitrogen-fixing Arcobacter or its descendant.

[0014] The preparation method of the microbial agent specifically comprises inoculating the A. shizayakii or its descendant and / or the nitrogen-fixing Arcobacter or its descendant into a liquid medium for culture, collecting a fermentation broth containing bacterial cells, centrifuging the fermentation broth containing a single strain or mixed strains to collect bacterial cell precipitates, and mixing the bacterial cell precipitates with biochar, potassium fulvate, bran, calcium carbonate, and diatomite, and then drying at room temperature to obtain a single microbial agent containing a single microorganism or a composite microbial agent containing two microorganisms.

[0015] Further, the liquid medium is a medium suitable for the growth of the A. shizayakii or its descendant and the nitrogen-fixing Arcobacter or its descendant.

[0016] Still further, the medium for culturing the A. shizayakii is an A. shizayakii isolation medium, which is composed of a solvent and solutes, the solvent being water, and each solute and its concentration in the medium being glucose 50.0 g / L, yeast extract 10.0 g / L, calcium carbonate 20.0 g / L, anhydrous ethanol 20-40 mL / L (final concentration 2-4%, added after separate filtration and sterilization), and Tween 80 1.0 g / L.

[0017] Further, the culture medium for culturing the Azotobacter is a nitrous oxide degrading bacteria isolation medium; the nitrous oxide degrading bacteria isolation medium is composed of a solvent and solutes, the solvent is water, and the solutes and their concentrations in the culture medium are as follows: KH2PO4 0.5 g / L, Na2HPO4·7H2O 1.0 g / L, MgSO4·7H2O 0.2 g / L, sodium succinate 2.0 g / L, N2O 5-10% (v / v), and trace element mother liquor 1 ml / L. The trace element mother liquor is composed of a solvent and solutes, the solvent is water, and the solutes and their concentrations in the culture medium are as follows: Na2EDTA 50 g / L, MnCl2·4H2O 5.06 g / L, ZnSO4·7H2O 2.2 g / L, CuSO4·5H2O 1.57 g / L, CaCl2 5.5 g / L, NaMoO4·4H2O g / L, FeSO4·7H2O 5.0 g / L, and CoCl2·6H2O 1.61 g / L. The fermentation culture conditions are as follows: 28°C, 180-200 rpm / min, and 36-48 h.

[0018] Further, the bacterial liquid containing single strain or mixed strains is centrifuged, the centrifugal speed can be 5000 rpm / min, and the centrifugal time can be 30 min.

[0019] Further, in the microbial agent, the mass percentage of the microbial precipitate in the microbial solid agent is 20% (dry matter accounts for about 6%), the mass percentage of the biochar in the microbial solid agent is 50%, the mass percentage of the potassium fulvate in the microbial solid agent is 14%, the mass percentage of the bran in the microbial solid agent is 20%, the mass percentage of the calcium carbonate in the microbial solid agent is 3%, and the mass percentage of the diatomite in the microbial solid agent is 7%.

[0020] In a fifth aspect, the present application provides an application of the above-mentioned composition or the above-mentioned Zymobacterium rhamnomyces or its offspring or the above-mentioned Azotobacter or its offspring, and the application is at least one selected from A1) to A6): A1) denitrogenation or nitrogen removal or nitrogen fixation or nitrogen-containing gas emission reduction; A2) preparation of a product for denitrogenation or nitrogen removal or nitrogen fixation or nitrogen-containing gas emission reduction; A3) removal of nitrogen-containing gas in waste; A4) preparation of a product for removal of nitrogen-containing gas in waste; A5) removal of nitrogen-containing gas generated in the fermentation process of organic materials or organic waste; A6) preparation of a product for removal of nitrogen-containing gas generated in the fermentation process of organic materials or organic waste.

[0021] The application as described above, wherein the nitrogen-containing gas is at least one of ammonia (NH3), nitrous oxide (N2O), nitric oxide (NO), nitrogen dioxide (NO2), gaseous nitrous acid (HONO).

[0022] The application as described above, wherein the organic material or organic waste includes at least one of livestock bedding, livestock manure.

[0023] In one specific embodiment, the complex microbial agent can be added to the organic material or organic waste for nitrogen-containing gas emission reduction.

[0024] In a sixth aspect, the present application provides a method for reducing the emission of nitrogen-containing gas in a sample, wherein the sample to be treated is contacted with any of the compositions described above or the Zymobacterium more than or its progeny or the Azotobacterium more than or its progeny, thereby reducing the emission of nitrogen-containing gas in the sample to be treated.

[0025] The method as described above, wherein the sample to be treated includes at least one of livestock bedding, livestock manure.

[0026] The present application can be used for nitrogen-containing gas emission reduction in bedding in a farm or in the process of livestock manure (normal, composting) treatment, and can effectively reduce the emission of ammonia, nitrous oxide, nitric oxide, nitrogen dioxide and gaseous nitrous acid. The microbial agent of the present application has significant emission reduction effect, is safe and harmless, and will play an important role in the process of nitrogen-containing gas emission reduction in bedding in a farm or in the process of nitrogen-containing gas emission reduction in solid organic waste such as livestock manure, and has good application prospect.

[0027] Deposit Description Strain name: Zymobacterium more than Acetobacter oryzae ) Strain number: RO Preservation agency: China General Microbiological Culture Collection Center Preservation agency abbreviation: CGMCC Address: No. 3, Beichen West Road, Beijing City, China Preservation date: October 13, 2025 Preservation center registration number: CGMCC No. 36149.

[0028] Deposit Description Strain name: Azoarcus sp. Strain number: YC Depositary: China General Microbiological Culture Collection Center Abbreviation of the depositary: CGMCC Address: No. 1, Yihuanli, Beijing, China Date of deposit: October 13, 2025 Center registration number: CGMCC No. 36150. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments. They should not be understood as limitations to the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present application. In the description of the present application, it should be understood that the terms used are only for the purpose of description and should not be understood as indicating or implying relative importance.

[0030] In the following examples, the experimental methods are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0031] In the quantitative test in the following examples, three repeated experiments were set up, and the average value was taken, unless otherwise specified.

[0032] In the following examples, the culture medium was configured as follows: Bacillus amyloliquefaciens isolation medium: glucose 50.0 g / L, yeast extract 10.0 g / L, calcium carbonate 20.0 g / L, anhydrous ethanol 20-40 mL / L (final concentration 2-4%, added after separate filtration and sterilization), Tween 80 1.0 g / L, solvent distilled water, pH natural.

[0033] Nitrous oxide degrading bacteria isolation medium: KH2PO4 0.5 g / L, Na2HPO4·7H2O 1.0 g / L, MgSO4·7H2O 0.2 g / L, sodium succinate 2.0 g / L, N2O proportion: 5-10% (v / v), trace element stock solution 1 ml / L, pH natural.

[0034] Trace element stock solution: Na₂EDTA 50 g / L, MnCl₂·4H₂O 5.06 g / L, ZnSO₄·7H₂O 2.2 g / L, CuSO₄·5H₂O 1.57 g / L, CaCl₂ 5.5 g / L, NaMoO₄·4H₂O g / L, FeSO₄·7H₂O 5.0 g / L, CoCl₂·6H₂O 1.61 g / L, pH natural.

[0035] After the above culture medium is prepared, it is sterilized at 121°C for 30 minutes.

[0036] The following examples used SPSS 16.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0037] Example 1, Acetobacter oryzae ( Acetobacter oryzae RO and nitrogen-fixing bacteria ( Azoarcus Isolation, purification, identification and preservation of sp.) YC I. Acetobacter oryzae ( Acetobacter oryzae Isolation, purification, identification and preservation of RO 1. Isolation and screening of strains The bacterial strain was isolated from the vinegar residue sample using conventional serial dilution plating. It was cultured on Acetobacter oryzae isolation medium at 28°C. Bacteria with significant differences in colony morphology and that could be cultured in pure form were selected, purified, and preserved on Acetobacter oryzae isolation medium. Finally, a bacterial strain was obtained and named strain RO.

[0038] 2. Identification of strains Morphological, physiological and biochemical characteristics, and some conserved sequences of strain RO were analyzed. Physiological, biochemical, and environmental tolerance characteristics were determined according to the methods described in "Principles and Methods of Soil Microbiology Research" (Lin Xiangui, Principles and Methods of Soil Microbiology Research [M], Higher Education Press, 2010).

[0039] (1) Morphological characteristics of strain RO: The bacterial cells of strain RO appear elliptical under a 400x optical microscope, arranged singly or in pairs. The cell surface is smooth, and no spore formation is observed. On Acetobacter oryzae isolation medium, this strain does not produce pigment, is opaque, and has a smooth or slightly rough surface. With prolonged culture time, the colonies gradually increase in size, with neat edges, a viscous texture, and are easily picked up by an inoculation loop.

[0040] (2) Physiological and biochemical characteristics of strain RO: Gram-negative, does not form a biofilm. Negative for methyl red, negative for acetylmethylethanol (VP), positive for nitrate reduction, negative for indole, positive for catalase, and negative for urase. It can oxidize ethanol to acetic acid, but cannot hydrolyze gelatin and starch. Negative for oxidase and does not produce hydrogen sulfide. It can utilize lactic acid, ethanol, and sucrose as carbon sources, but cannot utilize citrate and malonate. It produces acid from glucose, fructose, and galactose, but cannot utilize lactose or maltose. It cannot grow in carbon-free media.

[0041] (3) Environmental tolerance of strain RO: The environmental tolerance of strain RO is as follows: it can grow normally in media containing 1%-5% (mass content) sodium chloride, but cannot grow in media containing 7% (mass content) sodium chloride. It can grow normally in media with pH 4.0-7.0, with weak growth at pH 3.0 and no growth at pH 8.0. Growth is weak at 20℃, normal at 25℃-40℃, and the optimal growth temperature is 28℃.

[0042] (4) Molecular biological analysis: Genomic DNA was extracted from strain RO using a bacterial genomic DNA extraction kit, and the entire genome was sequenced. Based on the sequencing results, it was identified as *Acetobacter oryzae* (a type of bacteria). Acetobacter oryzae ).

[0043] Based on the above morphological characteristics, physiological and biochemical characteristics, and gene sequence analysis results, strain RO was identified as *Acetobacter oryzae* (also known as *Acetobacter oryzae*). Acetobacter oryzae ).

[0044] 3. Preservation of RO strains Strain RO was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36149. It will be referred to as Acetobacter oryzae CGMCC No. 36149 or strain RO.

[0045] II. Nitrosporium ( Acetobacter oryzae Isolation, purification, identification and preservation of sp.) YC 1. Isolation and screening of strains Bacterial strains were isolated from activated sludge samples in Beijing using conventional serial dilution plating. The strains were cultured on nitrous oxide-degrading bacteria isolation medium at 28°C. Bacteria with significant morphological differences and suitable for pure culture were selected and purified and preserved on nitrous oxide-degrading bacteria isolation medium. A single bacterial strain was finally obtained and named strain YC.

[0046] 2. Identification of strains Strain YC was analyzed for morphological, physiological and biochemical characteristics and partial conserved sequences. The physiological and biochemical characteristics and environmental tolerance were determined according to the methods described in Soil Microorganism Research Principles and Methods (Lin Xian- gui, Soil Microorganism Research Principles and Methods [M], Higher Education Press, 2010).

[0047] (1) Morphological characteristics of strain YC: The bacterial cells of strain YC were straight or slightly curved rods arranged singly or in pairs under a 400-fold optical microscope, and no spores were formed. The strain was motile. No pigment was produced on the nitrous oxide-degrading bacterial culture medium, and the colonies were colorless or milky white, semitransparent, round or subround, with an even edge, smooth surface and slight gloss. The colonies gradually increased in size, and the texture was moist or viscous as the culture time was prolonged.

[0048] (2) Physiological and biochemical characteristics of strain YC: The strain was gram-negative and could form a bacterial film in a liquid culture medium. The methyl red test was negative, the acetyl-methyl-methanol test (VP) was usually negative, the nitrate reduction test was positive, the indole reaction was negative, the contact enzyme was positive, the oxidase was positive, and the urine enzyme reaction varied with the strain. The strain could hydrolyze esculin, but most strains could not hydrolyze gelatin and starch. The strain could utilize citrate as the sole carbon source, but could not utilize malonate. The arginine dihydrolase was positive, and the lysine and ornithine decarboxylase was usually negative. The strain could not ferment glucose, sucrose, maltose and other sugars, but could grow by respiration using glucose, fructose, succinic acid, lactic acid, pyruvic acid and other carbon sources. The strain could not utilize mannitol and sorbitol.

[0049] Strain YC had strong environmental tolerance. The strain could grow normally in a culture medium containing 1%-5% (mass content) sodium chloride, grow slowly or be inhibited in a culture medium containing more than 5% sodium chloride. The strain could grow normally in a pH range of 5.5-9.5, grow weakly at pH 4.5, and could not grow at pH 10 or above. The strain could grow normally at an environmental temperature of 20°C-45°C, and the optimum growth temperature was 28°C. The strain could not grow normally after being treated at 80°C for 15 min.

[0050] (3) Molecular biology analysis The genomic DNA of strain YC was extracted by using a bacterial genomic DNA extraction kit, and the 16S rDNA of the strain was specifically amplified. After the amplification product was purified and sequenced, the results were compared with the NCBI database. The results showed that the 16S rDNA sequence of strain YC had the highest homology (>99.9%) with the corresponding sequence of Azotobacter sp., and the strain was preliminarily identified as Azotobacter sp. The 16S rDNA of Azotobacter sp. is specifically shown in SEQ ID NO: 1. Azoarcus Azoarcus ​​

[0051] SEQ ID NO: 1 is specified as follows:

[0052] Based on the above morphological characteristics, physiological and biochemical characteristics, and 16S rDNA gene sequence analysis results, strain YC was identified as *Azotobacter zonalis*. Azoarcus sp.).

[0053] 3. Nitrogenous toxoplasmosis ( Azoarcus Preservation of sp.) Nitrogenocide ( Azoarcus Azoarcus Azoarcus The strain YC was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36150. It is hereinafter referred to as *Azotobacter nigra* CGMCC No. 36150 or strain YC.

[0054] Example 2: Preparation of solid microbial inoculants 1. Preparation of Acetobacter oryzae CGMCC No. 36149 inoculum A single colony of *Acetobacter oryzae* CGMCC No. 36149 was picked and inoculated into *Acetobacter oryzae* isolation medium and cultured at 28°C with shaking at 180-200 rpm / min for 24 h. Then, 7.5 ml of the bacterial suspension was aseptically inoculated into a 250 ml Erlenmeyer flask containing 150 mL of medium and cultured at 28°C with shaking at 180-200 rpm / min for 36-48 h until the concentration reached 3.4 × 10⁻⁶. 8 CFU / ml was used to obtain a culture medium of Acetobacter oryzae CGMCC No. 36149. The culture medium was centrifuged at 5000 rpm / min for 30 min, and the precipitate was collected. The biomass of the strain in the precipitate was 4.8 × 10⁻⁶. 9 CFU / g, with a moisture content controlled at around 70%.

[0055] 2. Preparation of *Azotobacter nitrificans* CGMCC No. 36150 inoculum A single colony of *Azotobacter nitrosacchariformis* CGMCC No. 36150 was picked and inoculated into nitrous oxide-degrading bacteria isolation medium. The culture was incubated at 28°C with shaking at 180-200 rpm / min for 24 h. Then, 7.5 ml of the bacterial suspension was aseptically transferred to a 250 ml Erlenmeyer flask containing 150 mL of medium and incubated at 28°C with shaking at 180-200 rpm / min for 36-48 h, until the concentration reached 5.6 × 10⁻⁶. 9 CFU / ml was used to obtain a culture medium of *Aeromonas nitrogenosa* strain CGMCC No. 36150. The culture medium was centrifuged at 5000 rpm / min for 30 min, and the precipitate was collected. The biomass of *Aeromonas nitrogenosa* CGMCC No. 36150 in the precipitate was 6.7 × 10⁻⁶ CFU / ml. 10 CFU / g, with a moisture content controlled at around 70%.

[0056] 3. Preparation of single and complex microbial inoculants The precipitates of K. sake CGMCC No. 36149 and A. azotofaciens CGMCC No. 36150 collected in 1-2 were prepared into single and complex microbial inoculants. The precipitates of K. sake CGMCC No. 36149 or A. azotofaciens CGMCC No. 36150 were mixed with biochar, potassium fulvate, bran, calcium carbonate and diatomite respectively, and then naturally air-dried to obtain the final single microbial solid inoculants. The precipitates of K. sake CGMCC No. 36149 and A. azotofaciens CGMCC No. 36150 were mixed with biochar, potassium fulvate, bran, calcium carbonate and diatomite, and then naturally air-dried to obtain the final complex microbial solid inoculants. The dry weight percentage of single or complex precipitates in the microbial solid inoculants was 6%, and the mass ratio of K. sake CGMCC No. 36149 to A. azotofaciens CGMCC No. 36150 in the complex precipitates was 1:1. The mass percentage of biochar in the microbial solid inoculants was 50%, the mass percentage of potassium fulvate in the microbial solid inoculants was 14%, the mass percentage of bran in the microbial solid inoculants was 20%, the mass percentage of calcium carbonate in the microbial solid inoculants was 3%, and the mass percentage of diatomite in the microbial solid inoculants was 7%. The biomass of K. sake CGMCC No. 36149 or A. azotofaciens CGMCC No. 36150 in the single solid inoculants was 2.8 x 10 8 CFU / g and 4.0 x 10 9 CFU / g, respectively. The biomass of K. sake CGMCC No. 36149 and A. azotofaciens CGMCC No. 36150 in the complex solid inoculants was 1.4 x 10 8 CFU / g and 2.0 x 10 9 CFU / g, respectively.

[0057] Example 3, Nitrogen gas emission reduction effect of bedding in dairy farm The test point is located in the workshop of the South Gate Pilot Base of the Chinese Academy of Agricultural Sciences (longitude 116.106678, latitude 40.224254), and the test is carried out at room temperature. The bedding used in this test is taken from a dairy farm in Pinggu District, Beijing. The bedding production method of the farm is as follows: the cow dung scraped by the scraping board in the cowshed is subjected to dry-wet separation by a dry-wet separator, and the solid dung obtained is fermented in a fermentation tank for about half a month. During the fermentation period, the front period is ventilated by a fan, and the later period is statically stacked without ventilation. The test uses a plastic tank with a size of 120 cm x 90 cm x 34 cm as a simulated cow bed, and the prepared bedding is placed in the plastic tank. The filling thickness of the bedding is 20 cm, and there are 12 simulated cow beds. The test period is 15 days (April 15-29, 2025).

[0058] The microbial inoculant prepared in Example 2 is used as a nitrogen-containing gas eliminating agent, and four treatments are set respectively. 0.05% mass fraction of water is added as a control treatment (referred to as CK), 0.05% mass fraction of single microbial solid-state inoculant of Pediococcus cerevisiae CGMCC No. 36149 is added as RO treatment group (referred to as RO, the concentration is 2.8 x 10 8 CFU / g), 0.05% mass fraction of single microbial solid-state inoculant of Azotobacter sp. CGMCC No. 36150 is added as YC treatment group (referred to as YC, the concentration is 4.0 x 10 9 CFU / g), and 0.05% mass fraction of composite inoculant is added as composite treatment group (referred to as composite, the concentration of RO is 1.4 x 10 8 CFU / g, and the concentration of YC is 2.0 x 10 9 CFU / g). Each treatment is repeated three times, and different gas samples are collected on the 0th, 1st, 2nd, 3rd, 5th, 8th, 11th and 14th days of the experiment.

[0059] Ammonia (NH3) is determined by dynamic box-boric acid absorption titration method. The boric acid absorption-titration method of ammonia first uses a 2% concentration boric acid solution to collect the gas sample, so that the ammonia in the air is absorbed and fixed by boric acid. Then, a mixed indicator is added to the absorption liquid, and a standard solution of sulfuric acid with a known concentration is used for titration. When the color of the solution changes from green to light purple, it is the end point. According to the volume of the acid standard solution consumed and the total volume of the sampled gas, the concentration of ammonia in the air can be calculated.

[0060] Nitrogen oxides (NO and NO2) are obtained by static box gas bag sampling and offline detection. After 30 minutes of static box deduction, 500 mL of gas sample is collected into a gas bag with a syringe, diluted with 2500 mL of nitrogen, and then determined by a thermal electric 17i type nitrogen oxide analyzer.

[0061] Gaseous nitrous acid (HONO) was obtained by colorimetry after absorption by dynamic box method. HONO emission flux was determined by improved closed chamber intermittent pumping method. The calculation formula is as follows: Formula 1 In formula 1, is the HONO emission flux of the cow manure bedding, unit: ngN / m 2 ·s; and are the nitrite concentrations (calculated as NO2 - ) of the first channel and the second channel for determining the HONO emission of the cow manure bedding, unit: mg / L; and are the nitrite concentrations (calculated as NO2 - ) of the first channel and the second channel for determining the atmospheric HONO background, unit: mg / L; is the nitrogen concentration conversion coefficient, 14 / 46; is the absorption liquid volume, unit: L; 10 6 is the mass conversion coefficient; is the bottom surface area of the pumping chamber, unit: m 2 ; is the actual cumulative absorption HONO time, unit: h; 3.6x10 3 is the time conversion coefficient. The HONO monitoring parameters are shown in Table 1.

[0062] Table 1, HONO monitoring parameters

[0063] Greenhouse gas N2O static box method sampling tube collection, GC offline detection method. At 0, 5, 10, 15, 20 minutes after the static box, about 40 mL sample was injected into a 12 mL capacity glass sampling tube with a sampling pump, and the gas concentration was analyzed and tested by a gas chromatograph.

[0064] The whole experiment lasted for 14 days, and the detection of 5 kinds of gases and the calculation of the total amount of 5 kinds of gases were carried out at 0d, 1d, 2d, 3d, 5d, 8d, 11d and 14d of the experiment, respectively. The detection results are shown in Tables 2-7, and the addition of microbial preparation (RO, YC, and complex microbial agent) can effectively reduce the emission of nitrogen-containing gases. In particular, the emission reduction effect of the complex microbial agent is obviously better than that of single strain, which can significantly reduce the emission of 5 kinds of nitrogen-containing gases and the total amount of 5 kinds of gases.

[0065] Table 2, ammonia emission detection results

[0066] According to Table 2, during the entire detection process, the addition of biological bacterial agent can reduce the emission of ammonia gas, and the effect of composite bacterial agent is better. During the entire process, the composite bacterial agent can significantly reduce the emission of ammonia gas (compared with the control), the RO treatment significantly reduces the emission of ammonia gas after the 3rd day, and the YC treatment significantly reduces the emission of ammonia gas after the 1st day. On the 8th day of treatment, the ammonia emission reduction rate of the composite bacterial agent is 81.47%, which is higher than that of other treatments.

[0067] Table 3, Nitrogen oxide (NO) emission detection results

[0068] According to Table 3, the effect of composite bacterial agent on NO emission reduction is better than that of single strain. During the entire experiment, it significantly reduces the emission of NO, and on the 11th day, NO emission is not detected (100% reduction rate). YC treatment significantly reduces the emission of NO from the 3rd day.

[0069] Table 4, Nitrogen oxide (NO2) emission detection results

[0070] According to Table 4, the emission of NO2 is less during the experiment, and the emission decreases to zero on the 5th day. The effect of composite bacterial agent on NO2 emission reduction is better than that of single strain, and on the 1st day, NO2 is not detected (100% reduction rate).

[0071] Table 5, HONO emission detection results

[0072] According to Table 5, different bacterial agents can reduce the emission of HONO, and the effect of composite bacterial agent on HONO emission reduction is better than that of single strain. During the entire experiment, the composite bacterial agent can significantly reduce the emission of HONO, and on the 11th day, the emission is zero. The effect of YC treatment is better than that of RO treatment, and on the 1st day, the emission of YC treatment is significantly lower than that of the control.

[0073] Table 6, N2O emission detection results

[0074] According to Table 6, different bacterial agents can reduce the emission of N2O, and the effect of composite bacterial agent on N2O emission reduction is better than that of single strain. During the entire experiment, the composite bacterial agent can significantly reduce the emission of N2O, and on the 3rd day, the emission is reduced by more than 50%.

[0075] The emission detection results of single gas in Tables 2-6 are statistically analyzed, and the total emission of five gases is calculated, and the results are shown in Table 7.

[0076] Table 7, 5 gas emission test results (NH3, NO, NO2, N2O and HONO)

[0077] According to Table 7, different microbial agents can reduce the emission of the total amount of 5 kinds of gases, and the emission reduction effect of the composite microbial agent on the total amount of gases is better than that of the single strain. The composite microbial agent can significantly reduce the emission of the total amount of gases during the whole experiment process, and the emission reduction effect on the 14th day is up to 83.84%. The treatment effect of YC is better than that of RO, and the single strain treatment reaches the maximum emission reduction rate of 62.27% and 70.66% on the 5th day.

[0078] In summary, the single microbial agent and the composite microbial agent treatment of the present application can effectively reduce the generation of nitrogen-containing gases, and the composite microbial agent has the best effect, which can significantly reduce the generation of 5 kinds of gases. Compared with the control, the composite microbial agent significantly reduces the emission of 5 kinds of gases, and the emission reduction effect of the composite microbial agent is better than that of the single strain.

[0079] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A composition, characterized in that, This includes Acetobacter oryzae or its descendants and Azotobacter nitrificans or its descendants; The Acetobacter oryzae is deposited at the China General Microbiological Culture Collection Center (CGMCC), and its registration number at CGMCC is CGMCC No. 36149. The *Azotocinobacter* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), and its registration number at CGMCC is CGMCC No. 36150.

2. The composition according to claim 1, characterized in that, The composition is a microbial agent.

3. The composition according to claim 1 or 2, characterized in that, Based on each gram of the composition, the composition contains 1.0 × 10⁻⁶ 8 -8.0×10 8 CFU of Acetobacter oryzae or its progeny and 1.0 × 10 9 -1.0×10 10 CFU (Cytoxobacterium nitrificum) or its progeny.

4. Acetobacter oryzae or its progeny, characterized in that, The *Acetobacter oryzae* is deposited at the China General Microbiological Culture Collection Center (CGMCC), and its registration number at CGMCC is CGMCC No. 36149.

5. *Azotocinus* or its progeny, characterized in that, The *Azotocinobacter* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), and its registration number at CGMCC is CGMCC No. 36150.

6. A method for preparing a microbial agent, characterized in that, The step includes preparing a bacterial agent from the composition of any one of claims 1-3, or from the Acetobacter oryzae or its descendants as described in claim 4, or from the Azotobacter zoatoides or its descendants as described in claim 5.

7. The application of the composition according to any one of claims 1-3, or the *Acetobacter oryzae* or its progeny according to claim 4, or the *Azotobacter* or its progeny according to claim 5, characterized in that, The application is selected from at least one of A1)-A6): A1) Denitrification, nitrogen removal, nitrogen fixation, or reduction of nitrogen-containing gas emissions; A2) Prepare products for nitrogen removal, nitrogen fixation, or nitrogen-containing gas emission reduction; A3) Removal of nitrogen-containing gases from waste; A4) Prepare products that remove nitrogen-containing gases from waste; A5) Remove nitrogen-containing gases produced during the fermentation of organic materials or organic waste; A6) Prepare products that remove nitrogen-containing gases produced during the fermentation of organic materials or organic waste.

8. The application according to claim 7, characterized in that, The nitrogen-containing gas includes at least one of ammonia, nitrous oxide, nitric oxide, nitrogen dioxide, and gaseous nitrous acid.

9. The application according to claim 7 or 8, characterized in that, The organic materials or organic waste include at least one of livestock and poultry bedding materials and livestock and poultry manure.

10. A method for reducing nitrogen gas emissions in a sample, characterized in that, Contact the sample to be treated with the composition of any one of claims 1-3, or with Acetobacter oryzae or its progeny as described in claim 4, or with Azotobacter nitrosamine or its progeny as described in claim 5, to reduce the emission of nitrogen-containing gases in the sample to be treated.

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